First-generation Vectors Research Articles

Adenoviral Vector System: A Comprehensive Overview of Constructions, Therapeutic Applications and Host Responses.

Adenoviral vectors are crucial for gene therapy and vaccine development, offering a platform for gene delivery into host cells. Since the discovery of adenoviruses, first-generation vectors with limited capacity have evolved to third-generation vectors flacking viral coding sequences, balancing safety and gene-carrying capacity. The applications of adenoviral vectors for gene therapy and anti-viral treatments have expanded through the use of in vitro ligation and homologous recombination, along with gene editing advancements such as CRISPR-Cas9. Current research aims to maintain the efficacy and safety of adenoviral vectors by addressing challenges such as pre-existing immunity against adenoviral vectors and developing new adenoviral vectors from rare adenovirus types and non-human species. In summary, adenoviral vectors have great potential in gene therapy and vaccine development. Through continuous research and technological advancements, these vectors are expected to lead to the development of safer and more effective treatments.

Gutless Helper-Dependent and First-Generation HAdV5 Vectors Have Similar Mechanical Properties and Common Transduction Mechanisms.

Delivering vectorized information into cells with the help of viruses has been of high interest to fundamental and applied science, and bears significant therapeutic promise. Human adenoviruses (HAdVs) have been at the forefront of gene delivery for many years, and the subject of intensive development resulting in several generations of agents, including replication-competent, -defective or retargeted vectors, and recently also helper-dependent (HD), so-called gutless vectors lacking any viral protein coding information. While it is possible to produce HD-AdVs in significant amounts, physical properties of these virus-like particles and their efficiency of transduction have not been addressed. Here, we used single-cell and single virus particle assays to probe the effect of genome length on HAdV-C5 vector transduction. Our results demonstrate that first-generation C5 vectors lacking the E1/E3 regions of the viral genome as well as HD-AdV-C5 particles with a wild type (wt) ∼36 kbp or an undersized double-strand DNA genome are similar to human adenovirus C5 (HAdV-C5) wt regarding attachment to human lung epithelial cells, endocytic uptake, endosome penetration and dependency on the E3 RING ubiquitin ligase Mind Bomb 1 for DNA uncoating at the nuclear pore complex. Atomic force microscopy measurements of single virus particles indicated that small changes in the genome length from 94% to 103% of HAdV-C5 have no major impact on physical and mechanical features of AdV vectors. In contrast, an HD-AdV-C5 with ∼30 kbp genome was slightly stiffer and less heat-resistant than the other particles, despite comparable entry and transduction efficiencies in tissue culture cell lines, including murine alveolar macrophage-like Max-Planck-Institute (MPI)-2 cells. Together, our in vitro studies reinforce the use of HD-AdV vectors for effective single round gene delivery. The results illustrate how physical properties and cell entry behavior of single virus particles can provide functional information for anticipated therapeutic vector applications.

Third-generation rabies viral vectors allow nontoxic retrograde targeting of projection neurons with greatly increased efficiency

Rabies viral vectors have become important components of the systems neuroscience toolkit, allowing both direct retrograde targeting of projection neurons and monosynaptic tracing of inputs to defined postsynaptic populations, but the rapid cytotoxicity of first-generation (ΔG) vectors limits their use to short-term experiments. We recently introduced second-generation, double-deletion-mutant (ΔGL) rabies viral vectors, showing that they efficiently retrogradely infect projection neurons and express recombinases effectively but with little to no detectable toxicity; more recently, we have shown that ΔGL viruses can be used for monosynaptic tracing with far lower cytotoxicity than the first-generation system. Here, we introduce third-generation (ΔL) rabies viral vectors, which appear to be as nontoxic as second-generation ones but have the major advantage of growing to much higher titers, resulting in significantly increased numbers of retrogradely labeled neurons invivo.

The pComb3 Phagemid Family of Phage Display Vectors.

A phagemid is a plasmid that contains the origin of replication and packaging signal of a filamentous phage. Following bacterial transformation, a phagemid can be replicated and amplified as a plasmid, using a double-stranded DNA origin of replication, or it can be replicated as single-stranded DNA for packaging into filamentous phage particles. The use of phagemids enables phage display of large proteins, such as antibody fragments. Phagemid pComb3 was among the first phage display vectors used for the generation and selection of antibody libraries in the 50-kDa Fab format, a monovalent proxy of natural antibodies. Affording a robust and versatile tool for more than three decades, phage display vectors of the pComb3 phagemid family have been widely used for the discovery, affinity maturation, and humanization of antibodies in Fab, scFv, and single-domain formats from naive, immune, and synthetic antibody repertoires. In addition, they have been used for broadening phage display to the mining of nonimmunoglobulin repertoires. This review examines conceptual, functional, and molecular features of the first-generation phage display vector pComb3 and its successors, pComb3H, pComb3X, and pC3C.

Rescue of GM3 synthase deficiency by spatially controlled, rAAV-mediated ST3GAL5 delivery.

GM3 synthase deficiency (GM3SD) is an infantile-onset epileptic encephalopathy syndrome caused by biallelic loss-of-function mutations in ST3GAL5. Loss of ST3GAL5 activity in humans results in systemic ganglioside deficiency and severe neurological impairment. No disease-modifying treatment is currently available. Certain recombinant adeno-associated viruses (rAAVs) can cross the blood-brain barrier to induce widespread, long-term gene expression in the CNS and represent a promising therapeutic strategy. Here, we show that a first-generation rAAV-ST3GAL5 replacement vector using a ubiquitous promoter restored tissue ST3GAL5 expression and normalized cerebral gangliosides in patient-derived induced pluripotent stem cell neurons and brain tissue from St3gal5-KO mice but caused fatal hepatotoxicity when administered systemically. In contrast, a second-generation vector optimized for CNS-restricted ST3GAL5 expression, administered by either the intracerebroventricular or i.v. route at P1, allowed for safe and effective rescue of lethality and behavior impairment in symptomatic GM3SD mice up to a year. These results support further clinical development of ST3GAL5 gene therapy.

Preclinical assessment of an optimized AAV-FVIII vector in mice and non-human primates for the treatment of hemophilia A

Extensive clinical data from liver-mediated gene therapy trials have shown that dose-dependent immune responses against the vector capsid may impair or even preclude transgene expression if not managed successfully with prompt immune suppression. The goal of this preclinical study was to generate an adeno-associated viral (AAV) vector capable of expressing therapeutic levels of B-domain deleted factor VIII (FVIII) at the lowest possible vector dose to minimize the potential Risk of a capsid-mediated immune response in the clinical setting. Here, we describe the studies that identified the investigational agent SPK-8011, currently being evaluated in a phase 1/2 study (NCT03003533) in individuals with hemophilia A. In particular, the potency of our second-generation expression cassettes was evaluated in mice and in non-human primates using two different bioengineered capsids (AAV-Spark100 and AAV-Spark200). At 2 weeks after gene transfer, primates transduced with 2 × 1012 vg/kg AAV-Spark100-FVIII or AAV-Spark200-FVIII expressed FVIII antigen levels of 13% ± 2% and 22% ± 6% of normal, respectively. Collectively, these preclinical results validate the feasibility of lowering the AAV capsid dose for a gene-based therapeutic approach for hemophilia A to a dose level orders of magnitude lower than the first-generation vectors in the clinic.

Maternal COVID-19 Vaccination and Its Potential Impact on Fetal and Neonatal Development.

Vaccines have been developed at “warp speed” to combat the COVID-19 pandemic caused by the SARS-CoV-2 coronavirus. Although they are considered the best approach for preventing mortality, when assessing the safety of these vaccines, pregnant women have not been included in clinical trials. Thus, vaccine safety for this demographic, as well as for the developing fetus and neonate, remains to be determined. A global effort has been underway to encourage pregnant women to get vaccinated despite the uncertain risk posed to them and their offspring. Given this, post-hoc data collection, potentially for years, will be required to determine the outcomes of COVID-19 and vaccination on the next generation. Most COVID-19 vaccine reactions include injection site erythema, pain, swelling, fatigue, headache, fever and lymphadenopathy, which may be sufficient to affect fetal/neonatal development. In this review, we have explored components of the first-generation viral vector and mRNA COVID-19 vaccines that are believed to contribute to adverse reactions and which may negatively impact fetal and neonatal development. We have followed this with a discussion of the potential for using an ovine model to explore the long-term outcomes of COVID-19 vaccination during the prenatal and neonatal periods.

Capsid-Engineering for Central Nervous System-Directed Gene Therapy with Adeno-Associated Virus Vectors.

Closing the gap in knowledge on the cause of neurodegenerative disorders is paving the way toward innovative treatment strategies, among which gene therapy has emerged as a top candidate. Both conventional gene therapy and genome editing approaches are being developed, and a great number of human clinical trials are ongoing. Already 2 years ago, the first gene therapy for a neurodegenerative disease, spinal muscular atrophy type 1 (SMA1), obtained market approval. To realize such innovative strategies, gene therapy delivery tools are key assets. Here, we focus on recombinant adeno-associated virus (AAV) vectors and report on strategies to improve first-generation vectors. Current efforts focus on the viral capsid to modify the host-vector interaction aiming at increasing the efficacy of target cell transduction, at simplifying vector administration, and at reducing the risk of vector dose-related side effects.

Capsid Engineering Overcomes Barriers Toward Adeno-Associated Virus Vector-Mediated Transduction of Endothelial Cells

Endothelial cells (EC) are targets in gene therapy and regenerative medicine, but they are inefficiently transduced with adeno-associated virus (AAV) vectors of various serotypes. To identify barriers hampering efficient transduction and to develop an optimized AAV variant for EC transduction, we screened an AAV serotype 2-based peptide display library on primary human macrovascular EC. Using a new high-throughput selection and monitoring protocol, we identified a capsid variant, AAV-VEC, which outperformed the parental serotype as well as first-generation targeting vectors in EC transduction. AAV vector uptake was improved, resulting in significantly higher transgene expression levels from single-stranded vector genomes detectable within a few hours post-transduction. Notably, AAV-VEC transduced not only proliferating EC but also quiescent EC, although higher particle-per-cell ratios had to be applied. Also, induced pluripotent stem cell-derived endothelial progenitor cells, a novel tool in regenerative medicine and gene therapy, were highly susceptible toward AAV-VEC transduction. Thus, overcoming barriers by capsid engineering significantly expands the AAV tool kit for a wide range of applications targeting EC.

Past, present and future of haemophilia gene therapy: From vectors and transgenes to known and unknown outcomes.

Since the 1960s, the pace of innovation in haemophilia treatment has been fast and furious and occasionally with unintended consequences. As newer technologies are harnessed to better treat, and potentially cure, haemophilias A and B, an understanding of their underlying scientific principles and their benefits and risks are essential for all stakeholders. This review summarizes the starts and stops of introducing FVIII and FIX transgenes clinically, beginning 20years ago. Lessons from earlier nonclinical and clinical experiments have been utilized to improve vector selection, vector design, promoter/enhancer cis control regions and codon-optimized transgenes to trigger in vivo clinical FVIII and FIX levels in the near-normal to normal ranges. Many known and unknown questions remain, and some, based upon benefit and risk, should be answered during larger phase 3 clinical trials. Prior clinical outcomes in haemophilia trials have not been standardized, making between-trial comparisons difficult. Going forward, haemophilia gene therapy clinical trials should utilize a standard set of core outcomes, to facilitate comparisons to other gene- and protein-based therapies. These outcomes will be more important as the field moves beyond the first-generation gene therapies into more complex vectors that may address the shortcomings of first-generation vectors and offer greater benefits to the patient.

Gene Therapy Studies in Hemoglobinopathies: Successes and Challenges

Allogeneic hematopoietic stem cell transplantation (HSCT) from bone marrow or umbilical cord blood is currently the only curative treatment for hemoglobinopathies. However, only a small percentage of patients can undergo this procedure because (i) it can only be performed in specialized units, and (ii) the populations affected by these diseases are poorly represented in the international registry of HSC donors. Gene therapy using autologous HSC is a powerful alternative to allogeneic HSCT. It circumvents the need for a matched, unrelated donor and thus avoids the risk of graft-versus-host disease and graft rejection after HSCT. Furthermore, the conditioning regimen required to allow the engraftment of genetically modified cells does not include immunosuppressive drugs responsible for infectious complications. Hence, the gene therapy procedure can be performed by many pediatric and adult hematological units - even those with limited or no expertise of allogeneic HSCT. The absence of any treatment-related deaths in gene therapy trials to date and the increasing safety of this therapeutic approach are now enabling its rapid, broad application worldwide. Lentiviral vectors (LVs) fulfill the complex criteria required for a successful outcome in gene therapy for β-hemoglobinopathies. The use of self-inactivating (SIN) LVs (generated by the deletion of viral promoters/enhancers in the U3 region of the 3' long terminal repeat) has greatly improved vector safety. In view of the results obtained since 2007, SIN LVs vectors appear to be associated with a much lower risk of insertional mutagenesis than first-generation gamma retroviral vectors. Clinical trials of globin-expressing LVs are now under way at several sites in Europe (Paris and Milan) and the United States. The initial results are encouraging with regard to the achievement of transfusion-independence. Despite these promising results, some issues deserve further investigation (e.g. optimization of the conditioning regimen, mobilization protocol, vectors and transduction protocol). The objective of the conditioning regimen is to efficiently destroy the diseased hematopoietic stem cells (using toxic and/or depleting monoclonal antibodies) while avoiding off-target toxicity in other organs. This should protect the patient's fertility, and prevent mucositis and liver toxicity. Secondly, the characteristic dyserythropoeisis in the patients' bone marrow must also to be taken into account when seeking to obtain the requisite number of hematopoietic stem cells. The hypercellular erythroid bone marrow compartment and the microenvironment are key parameters in this setting. There is currently an intense, fruitful debate as to the best mobilizing factor for these patients. Furthermore, the characteristic inflammatory context in Hb SS patients should allow the use of anti-inflammatory molecules before harvesting. Lastly, genome editing and homologous recombination technologies have undergone spectacular developments over the last couple of decades. In view of the impressive progress recently reported for the gene addition strategy, offering gene editing approaches to patients affected by β-hemoglobinopathies would pose a number of ethical and medical questions. DisclosuresNo relevant conflicts of interest to declare.

Gene Therapy for X-Linked Severe Combined Immunodeficiency: Where Do We Stand?

More than 20 years ago, X-linked severe combined immunodeficiency (SCID-X1) appeared to be the best condition to test the feasibility of hematopoietic stem cell gene therapy. The seminal SCID-X1 clinical studies, based on first-generation gammaretroviral vectors, demonstrated good long-term immune reconstitution in most treated patients despite the occurrence of vector-related leukemia in a few of them. This gene therapy has successfully enabled correction of the T cell defect. Natural killer and B cell defects were only partially restored, most likely due to the absence of a conditioning regimen. The success of these pioneering trials paved the way for the extension of gene-based treatment to many other diseases of the hematopoietic system, but the unfortunate serious adverse events led to extensive investigations to define the retrovirus integration profiles. This review puts into perspective the clinical experience of gene therapy for SCID-X1, with the development and implementation of new generations of safer vectors such as self-inactivating gammaretroviral or lentiviral vectors as well as major advances in integrome knowledge.

Faster T-cell development following gene therapy compared with haploidentical HSCT in the treatment of SCID-X1

AbstractDuring the last decade, gene therapy via ex vivo gene transfer into autologous hematopoietic stem cells has emerged as a convincing therapy for severe combined immunodeficiency caused by ILR2G mutation (SCID-X1) despite the occurrence of genotoxicity caused by the integration of first-generation retroviral vectors. However, the place of gene therapy among the therapeutic armamentarium remains to be defined. We retrospectively analyze and compare clinical outcomes and immune reconstitution in 13 consecutive SCID-X1 patients having undergone haploidentical hematopoietic stem cell transplantation (HSCT) and 14 SCID-X1 patients treated with gene therapy over the same period at a single center level: the Necker Children's Hospital (Paris, France). Our results show a clear advantage in terms of T-cell development of gene therapy over HSCT with a mismatched donor. Patients treated with gene therapy display a faster T-cell reconstitution and a better long-term thymic output. Interestingly, this advantage of gene therapy vs haploidentical HSCT seems to be independent of the existence of clinical graft-versus-host disease in the latter condition. If data of safety are confirmed over the long term, gene therapy for SCID-X1 appears to be an equal, if not superior, alternative to haploidentical HSCT.

56. AAV Vector-Mediated CRISPR Attacks on Proviral HIV-1 DNA for Purging of Cellular Reservoirs

HIV, the human immunodeficiency virus, is highly successful as a viral pathogen due to its ability to persist in the infected host as an integrated proviral DNA within cellular reservoirs, particularly in resting CD4+ T cells and macrophages. Current antiretroviral combination therapy can control viral spread and viremia but cannot eradicate latent HIV genomes, raising an urgent need for alternative strategies for long-term remission or cure. We combined two powerful technologies, synthetic AAV vectors and CRISPR-mediated gene engineering, with the ultimate aim to eliminate integrated HIV from latently infected human cells. Therefore, we first screened a library of peptide display mutants based on 12 different AAV serotypes and identified novel capsids that mediate potent transduction of relevant HIV target cells, including primary human T-lymphocytes and macrophages. Our lead candidates – two peptide-modified variants of AAV9 or AAVrh.10 – were capable of transducing >65% of primary humanCD4+ T cells and were superior to all 12 wildtype AAV isolates. Concurrently, we created AAV vector genomes that express – alone or in combination – the two essential CRISPR components, i.e., the Cas9 protein and a g(uide)RNA directed against either the HIV-1 long terminal repeats (LTRs), or against viral structural genes. AAV/CRISPR vector efficiency and specificity were validated using a T7 endonuclease assay, showing successful Cas9-mediated induction of double-stranded DNA breaks at the specific HIV-1 target regions and subsequent error-prone repair in HeLaP4 cells with a stably integrated HIV-1 genome. To further potentiate the approach, we exploited a highly customizable self-complementary AAV vector backbone recently developed in our lab that permits multiplexing up to three gRNAs under control of the U6, H1 and 7SK promoters in a single vector particle. We engineered this template to co-express our best gRNAs against the two HIV-1 LTRs and the viral gag gene, and compared its potency with our different first-generation single gRNA vectors. In reporter cells carrying a GFP-encoding HIV-1 genome, simultaneous cleavage was indeed detected at three different sites (5’LTR, 3’LTR and gag), reaching an impressive 63.5% cutting efficiency (versus 36.6-50% for the single gRNA vectors) and demonstrating the power of these vectors to cleave HIV-1 proviral DNA. Finally, we will present results from our ongoing validation of proviral cleavage in human T-cell lines that are latently infected with HIV-1 as a physiologically relevant model of HIV-1 latency. The described experiments prove the possibility to target, cut and destroy integrated HIV-1 proviral DNA with AAV/CRISPR vectors in human cells, and therefore support the further development of our tools and approach into a novel clinical modality for purging of latent HIV-1 reservoirs.

459. CAR-Transduced T Cells Demonstrate Increased CTL Function

Infection with HIV-1 results in CD4+ T cell depletion and the subsequent loss of immune function results in AIDS. Although HAART lowers plasma viremia, it requires life-long drug therapy. However, elite controllers are able to exert control over viral replication in the absence of cART therapy by maintaining polyfunctional HIV-specific T cells with high avidity TCR binding that target CTL epitopes where escape mutants have reduced fitness.To mimic increase immunosurveillance of elite controllers and prevent viral replication in a non-controlled setting, we will transduce T cells with CD4-CAR vectors to bolster and redirect the “designer T cells” (dTc) towards an HIV-specific target. Chimeric antigen receptors (CARs) are fusion proteins of an extracellular antigenbinding domain linked to intracellular T cell signaling domains. In CD4-CARs, the CD4 extracellular domain binds directly to diverse HIV envelopes and redirects the CTL activity. Env/CD4 binding is independent of both the MHC restriction and Nef down regulation. As in elite controllers, CD4-CARs targets an essential viral function where mutations would lower viral fitness and make viral escape difficult to achieve. The first-generation CD4-CAR vector, with only the T-cell receptor zeta (TCRζ) intracellular domain, killed Env+ cells in vitro but failed to control viremia in clinical trials. Recent studies with anti-tumor CARs show that additional co-stimulatory signals (CD28 and 4-1BB) improved CTL function in vitro, in murine models, and showed objective tumor regression in clinical trials.We hypothesize that genetically modified T cells designed to increase CTL killing of HIV+ cells, to persist in vivo, and to prevent viral infection will provide durable viral suppression in vivo and eliminate the need for life-long drug therapy as modeled by elite controllers. To determine what signaling domains would be necessary to mimic this immune protection, we developed a series of CD4-CAR vectors with various intracellular signaling domains. To protect dTc from infection, we co-transduced cells with the membrane-associated C46 (maC46), a fusion inhibitor tethered to dTc, that binds HIV and blocks viral replication thereby providing a strong selective advantage to transduced cells. Therefore, we stimulated PBMCs with αCD3αCD28 and co-transduced T cells with the series of CD4-CAR and maC46 vectors. To evaluate the functional activity of the dTc, we measured 1) HIV Env-specific cytotoxicity, 2) cytokine production using IFN-γ ELIspot, and 3) CAR-specific proliferation of dTc. CD4-CAR T cells demonstrated Env+ specific killing, increased frequency of IFN-γ spot-forming cells, and CAR-specific proliferation.Our long-term goal, to adoptively transfer dTc with increased CTL function (cytokine production, proliferative capacity, viral suppression). The control of viremia in the absence of HAART would revolutionize HIV treatments for patients with AIDS.

111. Efficient Transduction in Neonatal Mice via Systemic Administration of an Adenovirus Vector

An adenovirus (Ad) vector is widely used as a gene delivery vector due to their various advantages. An Ad vector is applicable to innate genetic diseases, which are crucial targets of gene therapy. Patients of several innate genetic disorders often display symptoms in their early stage of life and are danger of death without any treatments. Radical treatments in infant ages are required in such patients. Gene therapy in neonates and infants is promising for such innate genetic disorders. In addition, although immune responses to an Ad vector hamper Ad vector-mediated transduction in adults, it is expected that such inhibitions do not occur in neonates and infants because of their immature immunity; however, transduction properties of Ad vectors in neonates and infants have not been fully examined. In this study, Ad vector-mediated transduction properties in neonatal mice were examined.A first-generation Ad vector containing a CMV promoter-driven firefly luciferase expression cassette was administered to newborn mice on the second day of life at a dose of 5.9×1011 infectious unit (IFU)/kg (this titer is equal to 1010 IFU/5-weeks old mice) via retro-orbital sinus. First, biodistribution of an Ad vector in neonatal mice was investigated 2 days after administration. As is the case with adult mice, the highest copy numbers of Ad vector genome were found in the liver among the organs. The liver also showed the highest levels of luciferase expression among the organs in neonates, although the transduction efficiency in the neonatal liver was approximately 5-fold lower than that in the adult liver. On the other hand, neonatal mice exhibited 10-fold higher levels of luciferase expression in the heart than adult mice. While approximately more than 1000-fold higher transduction efficiency was found in the adult liver, compared with the adult heart, the neonatal liver exhibited only 30-fold higher transduction efficiency than the neonatal heart. In order to achieve liver-specific transduction, a liver-specific promoter (a fusion promoter composed of apolipoprotein E enhancer, the hepatocyte control region, and human alpha 1-antitrypsin promoter) was incorporated into an Ad vector. The Ad vector containing the liver-specific promoter mediated almost 100-fold higher transduction efficiencies in the liver, compared with those in the other organs. Second, Ad vector-induced hepatotoxicity in neonatal mice was evaluated. Obvious elevation in serum alanine aminotransferase (ALT) activity in neonatal mice was not observed within 2 weeks following Ad vector administration, indicating that this dose of Ad vectors did not provoke significant hepatotoxicity in neonatal mice. In addition, while anti-Ad antibodies were strongly induced in the serum of adult mice on 21 days post Ad vector injection, almost background levels of anti-Ad antibodies were detected in neonatal mice. These results provide crucial information for gene therapy in neonates and infants.

Evaluation of Transduction Properties of an Adenovirus Vector in Neonatal Mice

In gene therapy for congenital disorders, treatments during neonate and infant stages are promising. Replication-incompetent adenovirus (Ad) vectors have been used in gene therapy studies of genetic disorders; however, the transduction properties of Ad vectors in neonates and infants have not been fully examined. Accordingly, this study examined the properties of Ad vector-mediated transduction in neonatal mice. A first-generation Ad vector containing a cytomegalovirus (CMV) promoter-driven luciferase expression cassette was administered to neonatal mice on the second day of life via retro-orbital sinus. The highest Ad vector genome copy numbers and transgene expression were found in the neonatal liver. The neonatal heart exhibited the second highest levels of transgene expression among the organs examined. There was an approximately 1500-fold difference in the transgene expression levels between the adult liver and heart, while the neonatal liver exhibited only an approximately 30-fold higher level of transgene expression than the neonatal heart. A liver-specific promoter for firefly luciferase expression conferred a more than 100-fold higher luciferase expression in the liver relative to the other organs. No apparent hepatotoxicity was observed in neonatal mice following Ad vector administration. These findings should provide valuable information for gene therapy using Ad vectors in neonates and infants.

Genomic analysis of Sleeping Beauty transposon integration in human somatic cells.

The Sleeping Beauty (SB) transposon is a non-viral integrating vector system with proven efficacy for gene transfer and functional genomics. However, integration efficiency is negatively affected by the length of the transposon. To optimize the SB transposon machinery, the inverted repeats and the transposase gene underwent several modifications, resulting in the generation of the hyperactive SB100X transposase and of the high-capacity “sandwich” (SA) transposon. In this study, we report a side-by-side comparison of the SA and the widely used T2 arrangement of transposon vectors carrying increasing DNA cargoes, up to 18 kb. Clonal analysis of SA integrants in human epithelial cells and in immortalized keratinocytes demonstrates stability and integrity of the transposon independently from the cargo size and copy number-dependent expression of the cargo cassette. A genome-wide analysis of unambiguously mapped SA integrations in keratinocytes showed an almost random distribution, with an overrepresentation in repetitive elements (satellite, LINE and small RNAs) compared to a library representing insertions of the first-generation transposon vector and to gammaretroviral and lentiviral libraries. The SA transposon/SB100X integrating system therefore shows important features as a system for delivering large gene constructs for gene therapy applications.

Differential contribution of adeno-associated virus type 2 Rep protein expression and nucleic acid elements to inhibition of adenoviral replication in cis and in trans.

The helper-dependent adeno-associated virus type 2 (AAV-2) exhibits complex interactions with its helper adenovirus. Whereas AAV-2 is dependent on adenoviral functions for productive replication, it conversely inhibits adenoviral replication, both when its genome is present in trans after coinfection with both viruses and when it is present in cis, as in the production of recombinant adenovirus (rAd)/AAV-2 hybrid vectors. The notion that AAV-mediated inhibition of adenoviral replication is due predominantly to the expression of the AAV-2 Rep proteins was recently challenged by successful Rep78 expression in a rAd5 vector through recoding of the Rep open reading frame (ORF). We closely analyzed the relative contributions of AAV-2 nucleic acid elements and Rep protein expression to the inhibition of adenoviral replication in both of the above scenarios. When present in cis, a sequence element in the 3' part of the rep gene, comprising only the AAV-2 p40 promoter and the AAV-2 intron sequence, which we termed the RIS-Ad, completely blocks adenoviral replication. p5/p19 promoter-driven Rep protein expression, on the other hand, only weakly inhibits rAd/AAV-2 vector propagation, and by inactivation of the RIS-Ad, it is feasible to generate first-generation rAd vectors expressing functional Rep proteins. The RIS-Ad plays no role in the inhibition of adenoviral replication in trans in a model closely mimicking AAV-2-Ad coinfection. In this case, expression of the Rep proteins is required, as well as the presence of an amplifiable inverted terminal repeat (ITR)-containing template. Thus, very different AAV-2 elements and mechanisms are involved in inhibition of adenoviral replication during rAd/AAV-2 vector propagation and after Ad-AAV coinfection. This is the first study to systematically compare the contributions of AAV-2 protein expression and AAV-2 nucleic acid elements to the inhibition of adenoviral replication in rAd/AAV-2 hybrid vector generation and in AAV-2-adenovirus coinfection. This study shows that the two inhibitory processes are very different with regard to AAV-2 functions and the mechanisms involved. Whereas inhibition of rAd/AAV-2 hybrid vector propagation mostly involves a 3' nucleic acid element in the rep gene, inhibition of an adenoviral genome in trans requires the Rep proteins and the AAV ITRs. These findings have important implications both for a basic understanding of the AAV replication cycle and for generation of rAd/AAV-2 hybrid vectors expressing the nonstructural and structural proteins of AAV-2.

Improving adenovirus vector-mediated RNAi efficiency by lacking the expression of virus-associated RNAs

Several studies have reported that short hairpin RNA (shRNA)-mediated RNA interference (RNAi) was competitively inhibited by the expression of adenovirus (Ad)-encoded small RNAs (VA-RNAs), which are expressed from a replication-incompetent Ad vector, as well as a wild-type Ad; however, it remained to be clarified whether an shRNA-expressing Ad vector-mediated knockdown was inhibited by VA-RNAs transcribed from the same Ad vector genome. In this study, we demonstrated that a lack of VA-RNA expression from the Ad vector leads to an increase in knockdown efficiencies of Ad vector-mediated RNAi. In the cells transduced with a first-generation Ad vector (FG-Ad) expressing shRNA (FG-Ad-shRNA), the copy numbers of shRNA and VA-RNAs incorporated into the RNA-induced silencing complex (RISC) was comparable. In contrast, higher amounts of shRNA were found in the RISC when the cells were transduced with an shRNA-expressing helper-dependent Ad (HD-Ad) vector, in which all viral genes, including VA-RNAs, were deleted (HD-Ad-shRNA), compared with FG-Ad-shRNA. HD-Ad vectors expressing shRNA against luciferase and p53 showed 7.4% and 37.3% increases in the knockdown efficiencies compared to the corresponding FG-Ad-shRNA, respectively, following in vitro transduction. Furthermore, higher levels of knockdown efficiencies were also found by the transduction with shRNA-expressing Ad vectors lacking VA-RNA expression (AdΔVR-shRNA) than by transduction with FG-Ad-shRNA. These results indicate that VA-RNAs expressed from an Ad vector inhibit knockdown by the shRNA-expressing Ad vector and that HD-Ad-shRNA and AdΔVR-shRNA are a powerful framework for shRNA-mediated knockdown.